Acute Respiratory Distress Syndrome (ARDS) affects almost a quarter million patients annually, and is responsible for 3.6 million hospital days. ARDS has a mortality rate approaching 40%, and its primary causes are pneumonia and sepsis. Central to the pathophysiology of this lung injury is a sustained inflammatory response, which leads to mitochondrial damage, alveolar epithelia injury, and contributes to the formation of edema and impairment of gas exchange across the alveolus. Clinical treatment for ARDS is largely supportive, and many interventions (e.g. T-cell receptor blockades, angiotensin II receptor antagonists, cytokine blocking antibodies or corticosteroids) have not improved outcomes in ARDS. Thus, there is an unmet need for new therapies to reduce the high morbidity and mortality associated with ARDS. Protein ubiquitination is the major protein processing pathways in cells by which ubiquitin (Ub) flags a targeted protein for degradation through the 26s proteasome or lysosome. It plays such a critical role in biological processes and its dysregulation leads to many diseases. Unfortunately, bacterial infection often disrupts the protein ubiquitination process. We and many other investigators have shown that infection or other inflammatory stimuli will alter the mRNA and protein levels of Ub E3 ligases, thus affecting the levels and functions of their target proteins. Thus, uncovering new Ub E3 ligase-based molecular pathways that contribute to lung injury provides unique opportunities to potentially devise new strategies to attenuate ARDS. We propose a systematic analysis of protein ubiquitination networks in ARDS, which has not been executed before. We have already identified several high value protein targets and laid out the experiment plans. These studies will add to the investigation into this exciting and critically important area of lung injury and inflammation. Specifically, we will focus on identifying novel molecular pathways that modulate the inflammatory cascade, mitochondria function/mitophagy, and DAMPs/inflammasome activation in the lung, and develop novel therapeutics for ARDS. In all three areas, we will systematically investigate how and which protein ubiquitination processes are dysregulated during lung injury. We will carry out state-of- the-art High-Throughput Screening (HTS) to identify the relevant E3 ligase/substrate pathways in the lung injury process. We will examine the process of protein ubiquitination using sophisticated biochemical tools, which will provide detailed mechanistic data such as substrate ubiquitination site and E3 ligase binding motif. Molecular and genetic approaches (such as Crispr/Cas9 gene editing and in vivo gene transfer) will be used to study the functions of E3 ligase/substrate in lung injury. We will also use our drug discovery expertise to develop small molecules for use in preclinical lung injury models. This proposal will lay the groundwork for futures studies involving the discovery of small molecules targeting protein ubiquitination pathways in ARDS.